Research of human mast cells has been constrained by the lack of human mast cell lines and the inability to grow human mast cells in vitro. The major goal of this proposal is to grow human mast cells in vitro using growth factors and carefully designed cell culture systems, which will, thereby, lead to a better understanding of human mast cell growth and differentiation. In preliminary studies, human recombinant interleukin (rIL-3) caused the growth of large numbers of histaminecontaining, toluidine blue positive, granulated cells (HCGC) in bone marrow cultures. HCGC in the culture of normal bone marrow were more similar to basophils than mast cells, whereas HCGC derived from bone marrow of a patient with systemic mastocytosis (SM), were more characteristic of mast cells. The specific aims of this proposal are as follows: 1) to examine the effects of human rIL-3 alone and in combination with human granulocyte macrophage colony-stimulating factor (rGM-CSF) on the growth of HCGC from normal and SM bone marrow and analyze HCGC in these cultures for characteristics of mast cells 2) to determine whether normal skin fibroblasts, fibroblasts from SM skin or neurofibroma tissue, both of which contain increased numbers of mast cells, or Schwann cells stimulate human mast cell growth and differentiation from bone marrow or HCGC. 3) to grow mast cells from the HL60 promyelocytic cell line by simultaneously forcing cell maturation either with vitamin D3 or retinoic acid and specifically directing differentiation to the mast cell lineage with human rIL-3 in the presence or absence of fibroblasts or Schwann cell monolayers. 4) to study the proliferative effects of human rIl-3, fibroblasts, and Schwann cells on mast cells isolated from tissue. Attempts will be made to isolate pure populations of mast cells obtained in vitro using sophisticated cell sorting techniques with flow cytometry. Techniques employed in these studies will include cell culture, tissue digestion, cell isolation, histochemical staining, histamine analysis, histamine release, flow cytometry, and electron microscopy. These co-culture experiments are particularly important since the cellular components of microenvironment, and specifically the fibroblast in the mouse, appear to be powerful determinants of mast cell phenotype. They offer exciting potential for a better understanding of interactions among mast cells and other cells in the tissue microenvironment. They will also provide the opportunity to perform the longer term research goal of investigating the potential role for human mast cells in the regulation of various immunologic responses, such as the production of IgE. A greater understanding of these important aspects of mast cell biology will offer important insights into the pathophysiology of mast cell-related diseases.